This application claims priority under 35 U.S.C. xc2xa7119 of German Application Ser. No. 199 56 820.0 filed Nov. 25, 1999.
The present invention concerns a method for the detection of nucleic acids on an analytical element which contains a sample application zone and a detection zone, the analytical element enabling liquid transport from the sample application zone to the detection zone, wherein a sample is applied to the sample application zone of the analytical element and the nucleic acids contained in the sample can be detected qualitatively in the detection zone by hybridization with a detection probe and also quantitatively, preferably by means of marker groups. In addition new analytical elements and reagent kits for the detection of nucleic acids are provided.
Nucleic acids such as amplification products from a PCR are currently detected for diagnostic applications by specific hybridization of the nucleic acid to be detected with a detection probe which is usually an oligonucleotide and a non-radioactive detection reaction using marker groups that can be present in the nucleic acids to be detected or/and in the detection probe. A variety of different methods can be used for this detection reaction. Thus the nucleic acids to be detected can be directly hybridized with a detection probe immobilized on a solid phase such as a microtitre plate (Amplicor Detection Kit, Roche Diagnostics, EP-A-0 420 260). Furthermore the nucleic acid to be detected can be pre-hybridized with a labelled detection probe and the complex that is formed in this process can be captured on a solid phase such as a microtitre plate (PCR-Detection Kit, Roche Diagnostics).
The use of test strips in diagnostic procedures has been known for a long period. Thus the detection of analytes using test strips is described in EP-B-0 186 799, EP-B-0 262 328 and EP-A-0 926 498 which primarily concern immunological test formats.
Jou et al. (Human Mutation 5 (1995), 86-93) describe a method for the detection of nucleic acids on chromatographic test strips in which hapten groups are introduced into the nucleic acid to be detected to enable an immunological immobilization of the nucleic acids to be detected on test strips by means of anti-hapten antibodies. Capture of nucleic acids to be detected by specific hybridization to immobilized detection probes on test strips is described for example in U.S. Pat. No. 5,310,650, EP-B-0 612 354 and Reinhartz et al. (Gene 13.6 (1993), 221-226) and Rule et al. (Clin. Chem. 42 (1996), 1206-1209).
However, the above-mentioned methods in which the detection of nucleic acids on test strips is described have a number of disadvantages. Thus the sensitivity decreases when the nucleic acid is immobilized by means of sandwich complexes. Increasing sensitivity for example by increasing the test volume is limited by the restricted incorporation of marker groups into the nucleic acids to be detected.
However, it is not possible to directly detect nucleic acids using the said methods. The complex between target nucleic acid and hybridization probe is firstly formed in a preliminary step. For this purpose the target nucleic acid is denatured (by heat treatment or by adding an alkaline solution), neutralized if necessary, the probe is added and hybridization conditions are set up. The actual detection on the test carrier can only occur after this preprocessing. Since this pre-processing involves the dosed addition of reagents and the use of apparatus e.g. heating devices, it is not possible to utilize the essential advantages of test strip-based methods such as low requirement for apparatus, simple handling, few operating steps and the ability to carry out the method by untrained persons. Furthermore these additional steps result in a risk of contamination which, in the case of detecting amplified nucleic acids, can lead to false-positive results.
Also in the case of methods which allow a direct hybridization of the nucleic acid to be detected to a hybridization probe on the analytical element, no sensitive test procedures are known in the prior art which do not require pre-processing of the nucleic acid to be detected so that in these procedures the nucleic acid cannot be detected directly or not be detected directly after an amplification process. In these procedures the target nucleic acid, which is usually present in a double-stranded form, is converted into a single-stranded conformation for the hybridization. Several methods have been described for this. Thus according to the well-known hybridization methods the nucleic acid is completely denatured, for example by heat treatment or by adding alkaline reagents, before it is applied to the analytical element. Enzymatic methods are also known which enable a specific digestion of one strand of the target nucleic acid.(Rule et al. Clinical Chemistry 42, page 1206-1209 (1996)).
Pre-processing can only be omitted when the target nucleic acids are already completely present as single strands. However, this usually already leads to difficulties when detecting ribosomal target nucleic acids because large regions of the native secondary structure of these single-stranded nucleic acids are in a double-stranded form (for example RRNA). A theoretical alternative would be amplification methods which allow a specific synthesis of only one nucleic acid strand. An example of this is asymmetric PCR. However, this method is considerably less efficient than conventional PCR methods due to the linear amplification, the sensitivity is severely limited and inadequate for the detection of analytes that are only present at a low concentration. Thus methods known in the prior art which allow a direct hybridization of the nucleic acid to be detected on the analytical element also have the same disadvantages that are found with the indirect detection methods.
A major advantage of the invention is that the previously described preprocessing of the nucleic acid to be detected can be omitted and that the nucleic acids can be applied directly to an analytical element or after an amplification process. The reagents on the analytical element that are preferably impregnated enable a complete or partial denaturation of the target nucleic acid on the analytical element which also enables hybridization of nucleic acids that are applied in a double-stranded conformation. The invention offers several possibilities for this. Thus denaturing reagents can be placed beforehand on the test carrier at a suitable concentration and preferably in a dry form in order to completely denature the nucleic acids. The target nucleic acid can be applied to the analytical element in a liquid form, for example contained in the PCR reaction solution.
After the reagents have been dissolved, the target nucleic acid is completely denatured. Since hybridization to the probe oligonucleotide cannot take place under these conditions, the chemical medium must be changed appropriately while the analyte is transported, preferably chromatographically, through the test carrier. This can on the one hand be achieved by suitable chromatography buffers or by reagents that have been placed downstream. Denaturation by means of a base and in particular NAOH and neutralization by means of an acid is preferred for this. Alternatively the chemical medium can be adjusted such that the double-stranded conformation of the target nucleic acid is destabilized (partially denatured) and hybridization to the probe oligonucleotide is enabled or facilitated. A change of the chemical medium during chromatography is unnecessary in this method variant. Preferred reagents for this embodiment are chaotropic salts and particularly preferably guanidinium thiocyanate (GuSCN).
In order to detect the target nucleic acids hybridized to the probe oligonucleotide, it is additionally necessary to label the complex. The marker groups can be incorporated into the amplified target nucleic acid by means of enzymatic incorporation during an optional amplification process that is carried out beforehand or be bound to the target nucleic acid by hybridizing an additional appropriately labelled probe oligonucleotide.
It was surprisingly found that when the method described above is used, target nucleic acids which are applied to the analytical element in a double-stranded form hybridize very effectively to the probe oligonucleotide despite the very short time that is available during chromatographic transport of the target nucleic acid over the analytical element and can be detected with high sensitivity.